I wrote a script for LIMMA about 9 months ago, which worked fine. Today ran it again (I've updated to the current R version) But got the following error message after this script: adf<-new("AnnotatedDataFrame",data=phenotypicdata) Error in getClass(Class, where = topenv(parent.frame())) : "AnnotatedDataFrame" is not a defined class The entire script is below. The offending script is in red. #Load libraries source("http://bioconductor.org/biocLite.R") library(limma) library(Biobase) #change directory to folder where files are (c:/limmadegenes) #Change to directory with original data files #read in expression data file and phenotypic data. #Note that row.names=1 means that row names are #in column 1, exprdata<-read.table("exprsData.txt", header=TRUE,sep="\t",row.names=1,as.is=TRUE,fill=TRUE,) class(exprdata) dim(exprdata) colnames(exprdata) head(exprdata) #printout too long to paste phenotypicdata<-read.table("phenotypicdata.txt",row.names=1,header=TRU E,sep="\t") class(phenotypicdata) #returns: [1] "data.frame" dim(phenotypicdata) #returns: [1] 28 2 colnames(phenotypicdata) #returns: [1] "Species" "Time" rownames(phenotypicdata) #Coerse exprdata into a matrix myexprdata<-as.matrix(exprdata) class(myexprdata) #[1] "matrix" rownames(myexprdata) colnames(myexprdata) #Coerse phenotypicdata into a data frame myphenotypicdata<-as.data.frame(phenotypicdata) rownames(myphenotypicdata) colnames(myphenotypicdata) #[1] "species" "time" summary(myphenotypicdata) all(rownames(myphenotypicdata)==colnames(myexprdata)) #[1] TRUE #Create annotated Data Frame adf<-new("AnnotatedDataFrame",data=phenotypicdata) #dim means: dimension of an object. dim(adf) #rowNames columnNames # 28 2 rownames(adf) #NULL #Create eset object eset<-new("ExpressionSet",exprs=myexprdata,phenoData=adf) #Read in targets file targets <- readTargets("targets.txt") targets # Set up character list defining your arrays, include replicates TS <- paste(targets$Species, targets$Time, sep=".") #This script returns the following: TS # Turn TS into a factor variable which facilitates fitting TS <- factor(TS) #This script returns the following design <- model.matrix(~0+TS) #write design object to text file write.table(design,file="design.txt",sep="\t",col.names=NA) colnames(design) <- levels(TS) #for eset put in your M values - see ?lmFit for object types fit <- lmFit(eset, design) #contrast matrix cont.matrix<-makeContrasts(s24vss0=s.24-s.0, s48vss24=s.48-s.24, s96vss48=s.96-s.48, c24vsc0=c.24-c.0, c48vsc24=c.48-c.24, c96vsc48=c.96-c.48, c0vss0=c.0-s.0, c24vss24=c.24-s.24, c48vss48=c.48-s.48, c96vss96=c.96-s.96, levels=design) write.table(cont.matrix,file="cont.matrix.txt",sep="\t",col.names=NA) #estimate the contrasts and put in fit2 fit2 <- contrasts.fit(fit, cont.matrix) fit2 <- eBayes(fit2) s24vss0<-topTable(fit2,coef="s24vss0",number=400,adjust.method="BH",p. value=1) write.table(s24vss0,file="s24vss0.txt",sep="\t") s48vss24<-topTable(fit2,coef="s48vss24",number=400,adjust.method="BH", p.value=1) write.table(s48vss24,file="s48vss24.txt",sep="\t") s96vss48<-topTable(fit2,coef="s96vss48",number=400,adjust.method="BH", p.value=1) write.table(s96vss48,file="s96vss48.txt",sep="\t") c24vsc0<-topTable(fit2,coef="c24vsc0",number=400,adjust.method="BH",p. value=1) write.table(c24vsc0,file="c24vsc0.txt",sep="\t") c48vsc24<-topTable(fit2,coef="c48vsc24",number=400,adjust.method="BH", p.value=1) write.table(c48vsc24,file="c24vsc48.txt",sep="\t") c96vsc48<-topTable(fit2,coef="c96vsc48",number=400,adjust.method="BH", p.value=1) write.table(c96vsc48,file="c96vsc48.txt",sep="\t") c0vss0<-topTable(fit2,coef="c0vss0",number=400,adjust.method="BH",p.va lue=1) write.table(c0vss0,file="c0vss0.txt",sep="\t") c24vss24<-topTable(fit2,coef="c24vss24",number=400,adjust.method="BH", p.value=1) write.table(c24vss24,file="c24vss24.txt",sep="\t") c48vss48<-topTable(fit2,coef="c48vss48",number=400,adjust.method="BH", p.value=1) write.table(c48vss48,file="c48vss48.txt",sep="\t") c96vss96<-topTable(fit2,coef="c96vss96",number=400,adjust.method="BH", p.value=1) write.table(c96vss96,file="c96vss96.txt",sep="\t") Thank-you Sally [[alternative HTML version deleted]]